Our research efforts this past year were focused on two subprojects, elucidation of the mechanisms underlying postsynaptic receptor regulation and the characterization of presynaptic proteins and vesicle trafficking in sensory cells of the inner ear. The first subproject involves a characterization of glutamate receptor synaptic organization and the regulation of synaptic glutamate receptor expression and trafficking in neurons. This past year significant progress has been made in understanding the delivery of NMDA receptors to the synapse and the assembly of AMPA receptors in neurons. Last year we reported that aberrant subunit assembly of AMPA receptors occurs in hippocampal neurons of mice lacking the GluR2 subunit. This is a critical subunit that controls calcium flux of the ion channel, and most AMPA receptors contain this subunit. We have previously shown that pyramidal cells of the hippocampus contain two populations of AMPA receptors, those made up of GluR1 and GluR2 and those made up of GluR2 and GluR3, showing that the neuron is capable of regulating the assembly of two distinct complexes and targeting them to different cellular locations. In the hippocampus of GluR2 deleted mice, we found that the GluR1 and GluR3 are now assembled together, with increasing amounts of homomeric GluR1 and GluR3. Based on immunogold electron microscopy, the amount of GluR1 at the synapse is significantly reduced, while the total amount of GluR1 is the same as that in the wild type. This suggests that homomeric GluR1 or GluR1/GluR3 receptors are not as effectively delivered to the synapse or are not as stable at the synapse. One explanation is that homomeric receptors or GluR1/GluR3 receptors are retained in the endoplasmic reticulum and not available for insertion into the plasma membrane. This seems to be unlikely since the amount of endoglycosidase H sensitive receptor does not change. Our results show that GluR2 is critical in establishing the correct composition of AMPA receptors and in the correct delivery of the receptor to the synapse. In the normal situation, the assembly of GluR1/GluR2 and GluR3/GluR2 complexes may depend on the availability of the GluR2 subunit. Thus, slight changes in the synthesis of this subunit, which is known to occur under some pathological conditions, could significantly change the types of AMPA receptors in the neuron. Since the NMDA receptor performs a critical function at the synapse, it is important to understand how the number and composition of receptors at the synapse are regulated. In addition to being at the synapse, some NMDA receptors are extrasynaptic where they may have functions distinct from those at the synapse. Last year we reported that over-expression of NR2 subunits, but not NR1 (splice variants with either C2 or C2? cassettes), increased the total number of NMDA receptors present on the cell surface of cultured cerebellar granule cells. Over-expression of NR1 or NR2 subunits did not change the number of synaptic NMDA receptors. These results show that extrasynaptic receptors are influenced simply by the amount of NR2 subunits produced, but that synaptic receptors are controlled by additional mechanisms. Cells transfected with NR2 from which the PDZ interacting domain was removed showed a change in the number of extrasynaptic receptors, but not synaptic receptors. This indicates that the PDZ interacting domain of NR2 is not required for extrasynaptic expression but is required for synaptic expression. We are also interested in the molecular mechanisms underlying the synaptic delivery of NMDA receptors. In our earlier studies we found that SAP102, an interacting partner of the NMDA receptor, was present throughout the neuron while its companion PDZ protein, PSD-95, was much more restricted to the synapse. This led to the suggestion that SAP102 may be associated with NMDA receptors that are being transported to and from the synapse. Addressing this hypothesis, we carried out a yeast two hybrid screen using the PDZ domain of SAP102 as bait. One of the interacting proteins was Sec8, a component of the exocyst. We have confirmed that Sec8 interacts with a complex of SAP102 and the NMDA receptor. This interaction begins in the endoplasmic reticulum, and disruption of the Sec8 interaction using a dominant negative construct blocks the delivery of the NMDA receptor to the plasma membrane in both heterologous cells and neurons. These results point to a critical role of the exocyst in the delivery of the NMDA receptors by way of an interaction with the PDZ protein. In our studies of the distribution of glutamate receptors and related proteins, we made the interesting observation that NMDA receptors are associated with attachment plaques between cerebellar granule cell dendrites in glomerular structures. These structures also contain PSD-95. Our second subproject addressed proteins associated with neurotransmitter release and vesicle trafficking in hair cells. Neurotransmitter release in hair cells differs from that which occurs at conventional synapses in a number of ways including the number of vesicles released, the sustainability of the release, and the presence of a presynaptic body thought to facilitate vesicle positioning on the plasma membrane. As we reported last year, to identify novel proteins that may be involved in release of neurotransmitter from hair cells, we carried out a yeast two hybrid screen using the SNARE proteins, syntaxin 1, VAMP1 and SNAP-25 as baits. We identified a number of proteins and have reported on the characterization of two, ocsyn and SIP30, this past year. Ocsyn is a syntaxin interacting protein that is concentrated in a novel structure in the apical part of inner hair cells. Using a series of markers to intracelluar organelles, we conclude that ocsyn is associated with the subapical compartment, a specialized form of recycling endosome found in polarized cells and that it plays a role in organizing SNARE proteins on intracellular membranes in the apical region of hair cells. SIP30 is a SNAP25 interacting protein that has a widespread distribution and is found in many tissues but is especially abundant in brain.